Polynucleotide vaccine for papillomavirus

ABSTRACT

DNA constructs encoding papilloma virus gene products, capable of being expressed upon direct introduction into animal tissues, are novel prophylactic pharmaceuticals which can provide immune protection against infection by papilloma virus.

CROSS-RELATED TO OTHER APPLICATION

This is a continuation-in-part of U.S. Ser. No. 08/268,424 filed Jun.30, 1994, now abandoned.

FIELD OF THE INVENTION

This invention relates to the production and use of a novelpharmaceutical product: a nucleic acid which, when directly introducedinto living vertebrate tissue, induces an immune response whichspecifically recognizes papilloma virus.

BACKGROUND OF THE INVENTION

Papilloma virus (PV) infections occur in a variety of animals, includinghumans, sheep, dogs, cats, rabbits, monkeys, snakes and cattle.Papilloma viruses infect epithelial cells, generally inducing benignepithelial or fibroepithelial tumors at the site of infection. Papillomaviruses are species specific infective agents; e.g., a humanpapillomavirus generally does not infect a nonhuman animal.

Papilloma viruses may be classified into distinct groups based on thehost that they infect. Human papilloma viruses (HPV) are furtherclassified into more than 60 types based on DNA sequence homology (for areview, see Papilloma Viruses and Human Cancer, H. Pfister (ed.), CRCPress, Inc., 1990). Papilloma virus infections appear to inducetype-specific immunogenic responses in that a neutralizing immunity toinfection to one type of papilloma virus may not confer immunity againstanother type of papilloma virus.

In humans, different HPV types cause distinct diseases. HPV types 1,2,3, 4, 7, 10 and 26-29 cause benign warts in both normal andimmunocompromised individuals. HPV types 5, 9, 9, 12, 14, 15, 17, 19-25,36 and 46-50 cause flat lesions in immunocompromised individuals. HPVtypes 6, 11, 34, 39, 41-44 and 51-55 cause nonmalignant condylomata ofthe genital tract. HPV types 16 and 18 cause epithelial dysplasia of thegenital tract and are associated with the majority of in situ andinvasive carcinomas of the cervix, vagina, vulva and anal canal.

Immunological studies in animal systems have shown that the productionof neutralizing antibodies to papilloma virus antigens preventsinfection with the homologous virus. The development of effective humanpapilloma virus vaccines has been slowed by the inability to cultivatepapilloma viruses in vitro. The development of an effective HPV vaccinehas been particularly slowed by the absence of a suitable animal hostfor the direct study of HPV.

Neutralization of papilloma virus by antibodies appears to betype-specific and dependent upon conformational epitopes on the surfaceof the virus.

Papilloma viruses are small (50-60 nm), nonenveloped, icosahedral DNAviruses that encode for early and late genes. The open reading frames(ORFs) of the virus genomes are designated E1 to E7 and L1 and L2, where"E" denotes early and "L" denotes late. L1 and L2 encode virus capsidproteins. E1 to E3 and E5 to E7 are associated with functions such asviral replication and transformation.

The L1 protein is the major capsid protein and has a molecular weight of55-60K. L2 protein is a minor capsid protein which has a predictedmolecular weight of approximately 55K and an apparent molecular weightof 75-100K as determined by polyacrylamide gel electrophoresis. Electronmicroscopic and immunologic data suggest that most of the L2 protein isinternal to the L1 protein. The L2 proteins are highly conserved amongdifferent papilloma viruses, especially the 10 basic amino acids at theC-terminus. The L1 ORF is highly conserved among different papillomaviruses.

The L1 and L2 genes have been used to generate recombinant proteins forpotential use in the prevention and treatment of papilloma virusinfections. Zhou et al. cloned HPV type 16 L1 and L2 genes into avaccinia virus vector and infected CV-1 mammalian cells with therecombinant vector to produce virus-like particles (VLP). These studieswere interpreted as establishing that the expression of both HPV type 16L1 and L2 proteins in epithelial cells is necessary and sufficient toallow assembly of VLP. The expression of L1 protein alone or L2 proteinalone or double infection of cells with single recombinant vacciniavirus vectors containing L1 and L2 genes did not produce particles.

Bacterially-derived recombinant bovine papilloma virus L1 and L2 havebeen generated. Neutralizing sera to the recombinant bacterial proteinscross-reacted with native virus at low levels, presumably due todifferences in the conformations of the native and bacterially-derivedproteins.

Recombinant baculoviruses expressing HPV16 L1 or HPV16 L2 ORF have beenused to infect insect SF9 cells and produce recombinant L1 and L2proteins. Western blot analyses showed that the baculovirus-derived L1and L2 proteins reacted with antibody to HPV16. The production of HPV 16L1 and HPV16 L2 proteins by recombinant strains of Saccharomycescerevisiae has also been demonstrated.

Since cytotoxic T-lymphocytes (CTLs) in both mice and humans are capableof recognizing epitopes derived from conserved internal viral proteinsand are thought to be important in the immune response against viruses,efforts have been directed towards the development of CTL vaccinescapable of providing heterologous protection against different viralstrains.

It is known that CD8⁺ CTLs kill virally-infected cells when their T cellreceptors recognize viral peptides associated with MHC class Imolecules. These peptides are derived from endogenously synthesizedviral proteins, regardless of the protein's location or function withinthe virus. Thus, by recognition of epitopes from conserved viralproteins, CTLs may provide cross-strain protection. Peptides capable ofassociating with MHC class I for CTL recognition originate from proteinsthat are present in or pass through the cytoplasm or endoplasmicreticulum. Therefore, in general, exogenous proteins, which enter theendosomal processing pathway (as in the case of antigens presented byMHC class II molecules), are not effective at generating CD8⁺ CTLresponses.

Efforts to generate CTL responses have included the use of replicatingvectors to produce the protein antigen within the cell or have focusedupon the introduction of peptides into the cytosol. Both of theseapproaches have limitations that may reduce their utility as vaccines.Retroviral vectors have restrictions on the size and structure ofpolypeptides that can be expressed as fusion proteins while maintainingthe ability of the recombinant virus to replicate, and the effectivenessof vectors such as vaccinia for subsequent immunizations may becompromised by immune responses against the vectors themselves. Also,viral vectors and modified pathogens have inherent risks that may hindertheir use in humans. Furthermore, the selection of peptide epitopes tobe presented is dependent upon the structure of an individual's MHCantigens and, therefore, peptide vaccines may have limited effectivenessdue to the diversity of MHC haplotypes in outbred populations.

Intramuscular inoculation of polynucleotide constructs. i.e., DNAplasmids encoding proteins, have been shown to result in the in situgeneration of the protein in muscle cells. By using cDNA plasmids thatencode viral proteins, antibody responses that provide homologousprotection against subsequent challenge can be generated. The use ofpolynucleotide vaccines (PNVs) to generate antibodies may result in anincreased duration of the antibody responses as well as the provision ofan antigen that may have the proper post-translational modifications andconformation of the native protein (vs. a recombinant protein). Theviral proteins produced in vivo after PNV immunization may assume theirnative conformation, thereby eliciting the production of virusneutralizing antibody. The generation of CTL responses by this meansoffers the benefits of cross-strain protection without the use of a livepotentially pathogenic vector or attenuated virus.

Benvenisty et al. reported that CaCl₂ precipitated DNA introduced intomice intraperitoneally, intravenously or intramuscularly could beexpressed. More recently, intramuscular (i.m.) injection of DNAexpression vectors in mice was reported to result in the uptake of DNAby the muscle cells and expression of the protein encoded by the DNA (J.A. Wolff et al., 1990; G. Ascadi et al., 1991). The injected plasmidswere shown to be maintained extrachromosomally and did not replicate.Subsequently, persistent expression after i.m. injection in skeletalmuscle of rats, fish and primates, and cardiac muscle of rats has beenreported. The technique of using nucleic acids as immunogenic agents wasreported in WO90/11092 (4 Oct. 1990), in which naked polynucleotideswere used to vaccinate vertebrates.

The method is not limited to intramuscular injection. For example, theintroduction of gold microprojectiles coated with DNA encoding bovinegrowth hormone (BGH) into the skin of mice resulted in production ofanti-BGH antibodies in the mice. A jet injector has been used totransfect skin, muscle, fat, and mammary tissues of living animals.Various methods for introducing nucleic acids were reviewed by Donnelly,Ulmer and Liu (The Immunologist, 2:20, 1994).

This invention contemplates a variety of methods for introducing nucleicacids into living tissue to induce expression of proteins. Thisinvention provides methods for introducing viral proteins into theantigen processing pathway to generate virus-specific CTLs andantibodies. Thus, the need for specific therapeutic agents capable ofeliciting desired prophylactic immune responses against viral pathogensis met for papilloma virus by this invention. Therefore, this inventionprovides DNA constructs encoding viral proteins of the human papillomavirus which encode induce specific CTLs and antibodies.

The protective efficacy of DNA vaccination against subsequent viralchallenge is demonstrated by immunization with non-replicating plasmidDNA encoding one or more of the above mentioned viral proteins. This isadvantageous since no infectious agent is involved, no assembly of virusparticles is required, and determinant selection is permitted.Furthermore, because the sequence of some of the gene products isconserved among various types of papilloma viruses, protection againstsubsequent challenge by a different type of papilloma virus that ishomologous to or heterologous to the strain from which the cloned geneis obtained is enabled.

SUMMARY OF THE INVENTION

DNA constructs encoding papilloma virus gene products, capable of beingexpressed upon direct introduction into animal tissues are novelprophylactic and therapeutic pharmaceuticals which can provide immuneprotection against infection by papilloma virus.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the virus neutralizing antibody response induced in rabbitsinjected with CRPV L1 DNA, or with a mixture of L1 and L2 DNA (y-axis),and the corresponding ELISA titers induced the same.

FIG. 2. Antibody responses of rabbits injected with L1 DNA. ELISA titersagainst L1 VLP of rabbits given a single immunization with anarbitrarily selected dosage of 1 mg of L1 DNA are shown. Rabbitsinjected with control DNA did not produce detectable antibodies againstL1 VLP.

FIGS. 3(A-B). Effect of absorption with L1 VLP on antiserum obtained byimmunization with L1 DNA. A, Normal serum, and immune serum absorbedwith native or denatured VLPs as in (15), were tested for virusneutralizing activity. The mean areas of condylomas on 3 challengesites, measured 7 weeks after challenge, are shown. B, Immune serum froma rabbit that had been injected with L1 DNA was serially absorbed threetimes with native (circles) or denatured (squares) L1 VLP expressed in arecombinant yeast (Saccharomyces cerevisiae) strain. After each serialabsorption, aliquots of serum then were assayed for antibody activityagainst baculovirus-derived L1 VLP by ELISA. The ELISA titer of theabsorbed material is plotted as a percentage of the original ELISA titerof unabsorbed serum.

FIGS. 4(A-B). ELISA responses in assays for anti-CRPV E2 (A) and CRPV E7(b) antibodies. The net reaction rate (rate for post dose 4 minus ratefor preimmune at the same dilution) in mOD/min is shown for eachindividual rabbit.

FIG. 5. Protective responses obtained in rabbits vaccinated with L1 DNAafter challenge with CRPV.

DETAILED DESCRIPTION OF THE INVENTION

DNA constructs encoding papilloma virus gene products, capable of beingexpressed upon direct introduction into animal tissues are novelprophylactic and therapeutic pharmaceuticals which can provide immuneprotection against infection by papilloma virus.

This invention provides polynucleotides which, when directly introducedinto a vertebrate animal such as cottontail rabbits and humans, induceexpression of encoded peptides within the tissues of the animal. Wherethe peptide is one that does not occur in that animal except duringinfections, such as proteins associated with papilloma virus (PV), theimmune system of the animal is activated to launch a protectiveresponse. Because these exogenous proteins are produced by cells of thehost animal, they are processed and presented by the majorhistocompatibility complex (MHC). This recognition is analogous to thatwhich occurs upon actual infection with the related organism. Theresult, as shown herein, is induction of immune responses which mayprotect against virulent infection.

As used herein, a polynucleotide is a nucleic acid that containsessential regulatory elements such that upon introduction into a livingvertebrate cell, is able to direct cellular machinery to producetranslation products encoded by the genes comprising the polynucleotide.There are many embodiments of the instant invention which those skilledin the art can appreciate from the specification. For example, differenttranscriptional promoters, terminators, carrier vectors or specific genesequences may be used.

This invention provides nucleic acids which when introduced into animaltissues in vivo induces the expression of the papilloma virus geneproduct. Thus, for example, injection of DNA constructs of thisinvention into the muscle of rabbit induces expression of the encodedgene products and elicits virus neutralizing antibodies. Upon subsequentchallenge with cottontail rabbit papilloma virus (CRPV), using doseswhich cause lesions on all control rabbits, animals injected with thepolynucleotide vaccine exhibit much reduced lesions. Thus, thisinvention discloses a vaccine useful in humans to prevent papillomavirus infections.

DNA constructs encoding papilloma viral proteins elicit protectiveimmune responses in animals. As will be described in more detail below,immune responses in animals have included virus neutralizing antibodyand protection from viral challenge in rabbits with homologous types ofpapilloma virus.

In one embodiment, the vaccine product will consist of separate DNAplasmids encoding, for example, the L1, L2, E2, E4 proteins of papillomavirus, either alone or in combination.

Anticipated advantages over other vaccines include but are not limitedto increased breadth of protection due to CTL responses, increasedbreadth of antibody, and increased duration of protection.

In one embodiment of the invention, the L1 or L2 or L1+L2 from HPV type6a, 6b, 11, 16 or 18 protein sequence, obtained from clinical isolates,is cloned into an expression vector. The vector contains a promoter forRNA polymerase transcription, and a transcriptional terminator at theend of the HPV coding sequence. Examples of promoters include but arenot limited to CMV. Examples of transcriptional terminators include butare not limited to BGH. In addition, to assist in preparation of thepharmaceutical, an antibiotic resistance marker expressed in E. coli isalso preferably included in the expression vector. Neomycin resistancegenes or any other pharmaceutically acceptable antibiotic resistancemarker may be used. Further, to aid in the high level production of thepharmaceutical by fermentation in prokaryotic organisms, it isadvantageous for the vector to contain an origin of replication and beof high copy number. A variety of commercially available prokaryoticcloning vectors provide these benefits. It is desirable to removenon-essential DNA sequences.

Therefore, this invention provides expression vectors encoding a PVprotein as an immunogen. The invention offers a means to inducecross-type protective immunity without the need for self-replicatingagents. In addition, immunization with DNA offers a number of otheradvantages. First, this approach to vaccination should be applicable totumors as well as infectious agents, since the CD8+CTL response isimportant for immunological intervention in both pathophysiologicalprocesses. Therefore, eliciting an immune response against a proteincrucial to the transformation process may be an effective means ofcancer protection or immunotherapy. Second, the generation of antibodiesagainst expressed proteins after injection of DNA encoding a viralprotein suggests that this technology provides a facile and effectivemeans of making antibody-inducing vaccines.

The ease of producing and purifying DNA constructs compares favorablywith that of traditional protein purification, which facilitates thegeneration of combination vaccines. Thus, multiple constructs, forexample constructs encoding L1 and L2 proteins of one or more types ofHPV, may be prepared, mixed and co-administered. Finally, becauseprotein expression may be maintained for a period of time following DNAinjection, the persistence of B- and T-cell memory may be enhanced,thereby engendering long-lived humoral and cell-mediated immunity.

The limitations of proposed HPV vaccines emphasize the need fordevelopment of more effective means for prevention of infection andamelioration of disease. Generation of an improved CTL response againsta conserved protein may provide significant long-term, cross-reactiveimmunity.

We have demonstrated protein expression from PNV constructs in rabbitsby detection of host immune response directed against CRPV antigens. Theresults of these animal experiments indicate that direct DNA injectionmay provide a method for protection of humans against HPV infection anddisease.

A range of doses is compared for immunogenicity in order to optimizeconcentrations for use. It is predictable that dosages of 10, 50, 100,and 200 μg of DNA are efficacious in man.

Human efficacy is shown in volunteers who receive HPV DNA vaccine. Thecomposition, dosage and administration regimens for the vaccine arebased on the foregoing studies. Clinical efficacy is shown by infectionrate, illness scores, and duration of illness. These clinical findingsare compared with laboratory evaluation of host immune response andviral detection in order to determine surrogate markers which correlatewith protection.

Molecular biology for preparing and purifying DNA constructs enable thepreparation of the DNA pharmaceuticals of this invention. While standardtechniques of molecular biology are sufficient for the production of theproducts of this invention, the specific constructs disclosed hereinprovide novel therapeutics which may produce cross-strain protection.

The amount of expressible DNA to be introduced to a vaccine recipientwill depend on the strength of the transcriptional and translationalpromoters used in the DNA construct, and on the immunogenicity of theexpressed gene product. In general, an immunologically orprophylactically effective dose of about 1 μg to 1 mg. and preferablyabout 10 μg to 300 μg is administered directly into muscle tissue.Subcutaneous injection, intradermal introduction, impression through theskin, and other modes of administration such as intraperitoneal,intravenous, or inhalation delivery are also contemplated. It is alsocontemplated that booster vaccinations are to be provided.

The polynucleotide may be naked, that is, unassociated with anyproteins, adjuvants or other agents which affect the recipient's immunesystem. In this case, it is desirable for the polynucleotide to be in aphysiologically acceptable solution, such as, but not limited to,sterile saline or sterile buffered saline. Alternatively, thepolynucleotide may be associated with liposomes, such as lecithinliposomes or other liposomes known in the art, as a DNA-liposomemixture, or the DNA may be associated with an adjuvant known in the artto boost immune responses, such as a protein or other carrier. Agentswhich assist in the cellular uptake of DNA, such as, but not limited to,calcium ions, viral proteins and other transfection facilitating agentsmay also be used to advantage. These agents are generally referred to astransfection facilitating agents and as pharmaceutically acceptablecarriers.

There are several advantages of immunization with a gene rather than itsgene product. One advantage is the relative simplicity with which nativeor nearly native antigen may be presented to the immune system. Anotheradvantage of polynucleotide immunization is the potential for theimmunogen to enter the MHC class I pathway and evoke a cytotoxic T cellresponse. Since polynucleotide immunization may elicit both humoral andcell-mediated responses, another advantage may be that it provides arelatively simple method to survey a large number of viral genes andviral types for the vaccine potential. Immunization by injection ofpolynucleotides also allows the assembly of multicomponent vaccines bymixing individual components.

As used herein, the term gene refers to a segment of nucleic acid whichencodes a discrete polypeptide. The term pharmaceutical, and vaccine areused interchangeably to indicate compositions useful for inducing immuneresponses. The terms construct, and plasmid are used interchangeably.The term vector is used to indicate a DNA into which genes may be clonedfor use according to the method of this invention.

Accordingly, one embodiment of this invention is a method for using PVgenes to induce immune responses in vivo, in a vertebrate such as amammal, including a human, which comprises:

a) isolating at least one PV gene,

b) linking the gene to regulatory sequences such that the gene isoperatively linked to control sequences which, when introduced into aliving tissue direct the transcription initiation and subsequenttranslation of the gene,

c) introducing the gene into a living tissue, and

d) optionally, boosting with additional PV gene.

Another embodiment of this invention may be a method for protectingagainst heterologous types of PV. This is accomplished by administeringan immunologically effective amount of a nucleic acid which encodes aconserved PV epitope.

In another embodiment of this invention, the polynucleotide vaccineencodes another PV protein, such as L1 or L2 or E1 through E7 orcombinations thereof.

In another embodiment of this invention, the DNA construct encodesproteins of HPV types 6a, 6b, 11, 16, or 18, wherein the DNA constructis capable of being expressed upon introduction into animal tissues invivo and inducing an immune response against the expressed product ofthe encoded HPV gene. Combinations comprising such constructs withpolynucleotides encoding other antigens, unrelated to HPV, arecontemplated by the instant invention.

Examples of HPV gene encoding DNA constructs include:

V1J-L1, V1J-L2, V1J-E1, V1J-E2, V1J-E3, V1J-E4, V1J-E5, V1J-E6, V1J-E7,V1J-E1i E4, V1J-E1 E4-L1, V1J-E2-C

In specific embodiments of this invention, the DNA construct encodesCRPV L1 protein, wherein the DNA construct is capable of being expressedupon introduction into animal tissues in vivo and inducing an immuneresponse against the expressed product of the encoded CRPV gene.Combinations comprising such constructs with polynucleotides encodingother antigens, unrelated to CRPV, are contemplated by the instantinvention.

Examples of CRPV gene encoding DNA constructs include:

V1J-L1, V1J-L2, V1J-E1, V1J-E2, V1J-E3, V1J-E4, V1J-E5, V1J-E6, V1J-E7,V1J-E1i E4, V1J-E1 E4-L1, V1J-E2-C.

Pharmaceutically useful compositions comprising the DNA may beformulated according to known methods such as by the admixture of apharmaceutically acceptable carrier. Examples of such carriers andmethods of formulation may be found in Remington's PharmaceuticalSciences. To form a pharmaceutically acceptable composition suitable foreffective administration, such compositions will contain an effectiveamount of the HPV DNA.

Therapeutic or diagnostic compositions of the invention are administeredto an individual in amounts sufficient to treat or diagnose PVinfections. The effective amount may vary according to a variety offactors such as the individual's condition, weight, sex and age. Otherfactors include the mode of administration. Generally, the compositionswill be administered in dosages ranging from about 1 microgram to about1 milligram.

The pharmaceutical compositions may be provided to the individual by avariety of routes such as subcutaneous, topical, oral and intramuscular.

The vaccines of the invention comprise HPV DNA that encode recombinantproteins of HPV that contain the antigenic determinants that induce theformation of neutralizing antibodies in the human host. Such vaccinesare also safe enough to be administered without danger of clinicalinfection; do not have toxic side effects; can be administered by aneffective route; are stable; and are compatible with vaccine carriers.

The vaccines may be administered by a variety of routes, such as orally,parenterally, subcutaneously or intramuscularly. The dosage administeredmay vary with the condition, sex, weight, and age of the individual; theroute of administration; and the type PV of the vaccine. The vaccine maybe used in dosage forms such as capsules, suspensions, elixirs, orliquid solutions. The vaccine may be formulated with an immunologicallyacceptable carrier.

The vaccines are administered in prophylactically or therapeuticallyeffective amounts, that is, in amounts sufficient to generate aimmunologically protective response. The effective amount may varyaccording to the type of PV. The vaccine may be administered in singleor multiple doses.

The methods of the present invention make possible the formulation ofmonovalent and multivalent vaccines for preventing PV infection. Usingthe methods, either monovalent or multivalent PV vaccines may be made.For example, a monovalent HPV type 16 vaccine may be made by formulatingDNA encoding HPV 16 L1 protein or L2 protein or L1+L2 proteins.Alternatively, a multivalent HPV vaccine may be formulated by mixing DNAencoding HPV L1 or L2 or L1+L2 proteins from different HPV types.

The DNA may be used to generate antibodies. The term "antibody" as usedherein includes both polyclonal and monoclonal antibodies, as well asfragments thereof, such as, Fv, Fab and F(ab)2 fragments that arecapable of binding antigen or hapten.

The PV DNA and antibodies of the present invention may be used toserotype HPV or CRPV infection and for HPV screening. The HPV and CRPVDNA and antibodies lend themselves to the formulation of kits suitablefor the detection and serotyping of HPV or CRPV. Such a kit wouldcomprise a compartmentalized carrier suitable to hold in closeconfinement at least one container. The carrier would further comprisereagents such as HPV DNA or anti-HPV antibodies suitable for detecting avariety of HPV types. The carrier may also contain means for detectionsuch as labeled antigen or enzyme substrates or the like.

The following examples are provided to further define the inventionwithout, however, limiting the invention to the particulars of theseexamples.

EXAMPLE 1

VECTORS FOR VACCINE PRODUCTION

A) V1: The expression vector V1 was constructed from pCMVIE-AKI-DHFR Y.Whang et al., J. Virol. 61, 1796 (1987)!. The AKI and DHFR genes wereremoved by cutting the vector with EcoR I and self-ligating. This vectordoes not contain intron A in the CMV promoter, so it was added as a PCRfragment that had a deleted internal Sac I site at 1855 as numbered inB. S. Chapman et al., Nuc. Acids Res. 19, 3979 (1991)!. The templateused for the PCR reactions was pCMVintA-Lux, made by ligating the HindIII and Nhe I fragment from pCMV6a120 see B. S. Chapman et al., ibid.,!which includes hCMV-IE1 enhancer/promoter and intron A, into the HindIII and Xba I sites of pBL3 to generate pCMVIntBL. The 1881 base pairluciferase gene fragment (Hind III-Sma I Klenow filled-in) from RSV-LuxJ. R. de Wet et al., Mol. Cell Biol. 7, 725, 1987! was cloned into theSal I site of pCMVIntBL, which was Klenow filled-in and phosphatasetreated.

The primers that spanned intron A are: ##STR1## The primers used toremove the Sac I site are: ##STR2##

The PCR fragment was cut with Sac I and Bgl II and inserted into thevector which had been cut with the same enzymes.

B) V1J EXPRESSION VECTOR

Our purpose in creating V1J was to remove the promoter and transcriptiontermination elements from our vector, V1, in order to place them withina more defined context, create a more compact vector, and to improveplasmid purification yields.

V1J is derived from vectors V1 and pUC19, a commercially availableplasmid. V1 was digested with SspI and EcoRI restriction enzymesproducing two fragments of DNA. The smaller of these fragments,containing the CMVintA promoter and Bovine Growth Hormone (BGH)transcription termination elements which control the expression ofheterologous genes was purified from an agarose electrophoresis gel. Theends of this DNA fragment were then "blunted" using the T4 DNApolymerase enzyme in order to facilitate its ligation to another"blunt-ended" DNA fragment.

pUC19 was chosen to provide the "backbone" of the expression vector. Itis known to produce high yields of plasmid, is well-characterized bysequence and function, and is of minimum size. We removed the entire lacoperon from this vector, which was unnecessary for our purposes and maybe detrimental to plasmid yields and heterologous gene expression, bypartial digestion with the HaeII restriction enzyme. The remainingplasmid was purified from an agarose electrophoresis gel, blunt-endedwith the T4 DNA polymerase, treated with calf intestinal alkalinephosphatase, and ligated to the CMVintA/BGH element described above.Plasmids exhibiting either of two possible orientations of the promoterelements within the pUC backbone were obtained. One of these plasmidsgave much higher yields of DNA in E. coli and was designated V 1J. Thisvector's structure was verified by sequence analysis of the junctionregions and was subsequently demonstrated to give comparable or higherexpression of heterologous genes compared with V1.

C) V1Jneo EXPRESSION VECTOR

It was necessary to remove the amp^(r) gene used for antibioticselection of bacteria harboring V1J because ampicillin may not be usedin large-scale fermenters for the production of human clinical products.The amp^(r) gene from the pUC backbone of V1J was removed by digestionwith SspI and Eam1105I restriction enzymes. The remaining plasmid waspurified by agarose gel electrophoresis, blunt-ended with T4 DNApolymerase, and then treated with calf intestinal alkaline phosphatase.The commercially available kan^(r) gene, derived from transposon 903 andcontained within the pUC4K plasmid, was excised using the PstIrestriction enzyme, purified by agarose gel electrophoresis, andblunt-ended with T4 DNA polymerase. This fragment was ligated with theV1J backbone and plasmids with the kan^(r) gene in either orientationwere derived which were designated as V1Jneo #'s 1 and 3. Each of theseplasmids was confirmed by restriction enzyme digestion analysis, DNAsequencing of the junction regions, and was shown to produce similarquantities of plasmid as V1J. Expression of heterologous gene productswas also comparable to V1J for these V1Jneo vectors. We arbitrarilyselected V1Jneo#3, referred to as V1Jneo hereafter, which contains thekan^(r) gene in the same orientation as the amp^(r) gene in V1J as theexpression construct.

D) V1Jns EXPRESSION VECTOR:

An Sfi I site was added to V1Jneo to facilitate integration studies. Acommercially available 13 base pair Sfi I linker (New England BioLabs)was added at the Kpn I site within the BGH sequence of the vector.V1Jneo was linearized with Kpn I, gel purified, blunted by T4 DNApolymerase, and ligated to the blunt Sfi I linker. Clonal isolates werechosen by restriction mapping and verified by sequencing through thelinker. The new vector was designated V1Jns. Expression of heterologousgenes in V1Jns (with Sfi I) was comparable to expression of the samegenes in V1Jneo (with Kpn I).

EXAMPLE 2

Preparation Of DNA Constructs Encoding Cottontail Rabbit Papilloma VirusProteins

The source of the CRPV DNA for all cloned genes is CRPV-pLAII. This isthe entire CRPV genome cloned into pBR322 at the Sal I site (Nasseri,M., Meyers, C. and Wettstein, F. O. (1989) Genetic analysis of CRPVpathogenesis: The L1 open reading frame is dispensable for cellulartransformation but is required for papilloma formation, Virology 170,321-325).

1. V1Jns-L1: The L1 coding sequence was generated by PCR, using theCRPV-pLAII DNA as template. The PCR primers were designed to contain BamHI sites for cleavage after the PCR fragment was gel purified.

The primers used to generate the L1 coding region were: ##STR3##

The PCR fragment was gel purified, cut with Bam HI and ligated to V1Jnscut with Bgl II.

2. V1Jns-L2: The L2 coding region was generated by PCR. The vectorCRPV-pLAII has the L2 gene disrupted by the Sal I site used in insertingCRPV into pBR322. Therefore, a template for PCR was generated by cuttingCRPV-pLAII with SalI and ligating the CRPV DNA into circular form at theSAl I site. This ligated CRPV DNA was used as the template for PCR. ThePCR primers were designed to contain Bam HI sites for cleavage after thePCR fragment was gel purified.

The primers used to generate the L2 coding region were: ##STR4##

3. V1Jns-E2: The E2 coding region is generated by PCR, using theCRPV-pLAII DNA as template. The PCR primers are designed to contain BglII sites for cleavage after the PCR fragment is gel purified.

The primers used to generate the E2 coding region are: ##STR5##

4. V1Jns-E4: The E4 coding region is generated by PCR, using theCRPV-pLAII DNA as template. The PCR primers are designed to contain BglII sites for cleavage after the PCR fragment is gel purified.

The primers used to generate the E4 coding region are: ##STR6##

5. V1Jns-E7: The E7 coding region was generated by PCR, using theCRPV-pLAII as template in one case and purified DNA from Kreider's CRPVstrain in another case. The same PCR primers were used for bothtemplates. The PCR primers are designed to contain Bgl II sites forcleavage after the PCR fragment was gel purified.

The primers used to generate the E7 coding region were: ##STR7##

6. pGEX-2T-E2: The E2 coding region was generated by PCR as describedfor V1Jns-E2. The fragment was cloned into pGEX-2T into the Bam HI siteto generate an in-frame fusion to glutathione S-transferase (GST). Thisconstruct is used to generate protein in E. coli.

7. pGEX-2T-E4: The E4 coding region was generated by PCR as describedfor V1Jns-E4. The fragment was cloned into pGEX-2T into the Barn HI siteto generate an in-frame fusion to glutathione S-transferase (GST). Thisconstruct is used to generate protein in E. coli.

8. pGEX-2T-E7: The E7 coding region was generated by PCR as describedfor V1Jns-E7. The fragment was cloned into pGEX-2T into the Bam HI siteto generate an in-frame fusion to glutathione S-transferase (GST). Thisconstruct is used to generate protein in E. coli.

EXAMPLE 3

Plasmid Purification from E. coli

V1J constructs were grown overnight to saturation. Cells were harvestedand lysed by a modification of an alkaline SDS procedure (Sambrook, J.,Fritsch, E. F., And Maniatis, T., Molecular Cloning: A LaboratoryManual. Cold Spring Harbor Laboratory Press. Cold Spring Harbor, N.Y.,ed.2 (1989). The modification consisted of increasing the volumesthree-fold for cell lysis and DNA extraction. DNA was purified by doublebanding on CsCl-EtBr gradients. The ethidium bromide was removed by1-butanol extraction. The resulting DNA was extracted withphenol/chloroform and precipitated with ethanol. DNA was resuspended inTE (10 mM Tris, 1 mM EDTA), pH 8 for transfections and in 0.9% NaCl forinjection into mice. Concentration and purity of each DNA preparationwas determined by OD_(260/280) readings. The 260/280 ratios were ≧1.8.

EXAMPLE 4

Production of CRPV Specific Antibodies In Vivo

Five rabbits per group were bled and then injected with 1.2 ml of salinecontaining 1 mg of V1Jns-L1, V1Jns-L2, V1Jns-L1 mixed with V1Jns-L2 (2mg total), or with V1Jns (control vector with no protein encoded) alone.The inoculum was divided equally among six intramuscular sites on bothhind legs, both forelegs, and the lower back. Three weeks after theinitial DNA injection, the rabbits were bled and given a secondinjection of the same DNA in the same manner. Four weeks after thesecond injection, the animals were bled again.

Sera were tested for virus neutralizing antibody by mixing tenfoldserial dilutions of immune serum with a 1:3 dilution of CRPV stock virus(Kreider strain). Dilutions were prepared in Dulbecco's Modified Eagle'sMedium (DMEM) supplemented with 1% bovine serum albumin (BSA). CRPVstock virus (purchased from Dr. J. Kreider, Hershey, Pa.) was preparedfrom skin fragments obtained from wild cottontail rabbits, which wereinfected with CRPV and implanted under the renal capsules of athymicmice. The resulting condylomas were homogenized and clarified bycentrifugation to yield a stock virus preparation. The mixtures ofimmune serum and virus stock were incubated on ice for at least 60minutes, and then 50 μl of each mixture was applied to a 1 cm² area ofshaved, scarified skin on the backs of 3 New Zealand White rabbits. Theanimals were observed 7 weeks later for the presence of warts and theanteroposterior and lateral dimensions (in mm) of the ellipsoidal wartswere measured. Endpoint titers were determined from the frequency ofwarts at the various dilutions by Reed-Muench interpolation.Neutralizing antibody titers of rabbits injected with L1 DNA or both L1and L2 DNA are plotted on the y-axis of FIG. 1.

The sera from rabbits that had been injected with L1 DNA also weretested for antibody by ELISA. Polystyrene ELISA plates were coatedovernight at 4° C. with 1 μg/well of semipurified, recombinantyeast-derived CRPV L1 protein. The recombinant L1 was prepared in S.cerevisiae and purified as described by Kirnbauer et al. (Proc. Nat.Acad. Sci. U.S.A. 89:12180-4, 1992) with minor modifications. Dilutedsera were added and incubated for 1 hour at room temperature (withshaking on an orbital shaker). The plates were then washed andhorseradish peroxidase-labeled goat anti-rabbit IgG (Fc specific) wasadded. After one hour of incubation with shaking the plates were washedand substrate was added. Plates were read at 450 nm using a kineticELISA reader (Molecular Devices Corp.), and the values obtained werecorrected for background by subtraction of the reaction rate of thepreimmune serum from that of the postimmnunization serum at the samedilution. Titers were determined by interpolation of the resulting curveof corrected reaction rate versus dilution to a rate value of 10mOD/min. ELISA titers of rabbits injected with L1 DNA or L1 plus L2 DNAare plotted on the x-axis of FIG. 1. FIG. 1 shows that 12/13 sera thatwere positive for neutralizing activity (log titer≧1, i.e., positivewith undiluted serum) also were positive for ELISA antibody (ELISAtiter≧100). Four of the 4 sera that were negative for neutralizingantibody had ELISA titers≦350. All of the sera from rabbits thatreceived either L2 DNA alone or the V1J control vector had ELISA titersof less than 100. Taken together, these data show that antibodiesspecific for CRPV and capable of neutralizing it were obtained afterinjection of L1 DNA. ELISA titers in rabbits persisted undiminished forat least 32 weeks following immunization (FIG. 2).

EXAMPLE 5

Protection of Rabbits upon Challenge with Virulent CRPV

Five rabbits per group were injected intramuscularly with 1 mg, ofV1Jns-L1, V1Jns-L2, V1Jns-L1 mixed with V1Jns-L2 (2 mg total), or withV1Jns (control vector with no protein encoded) alone, as describedabove. Three weeks after the initial DNA injection, the rabbits receiveda second injection of the same DNA. Four weeks after the secondinjection, the rabbits were challenged with CRPV. The CRPV challenge wasperformed by applying 50 μl of two dilutions of virus stock (diluted 1:2or 1:12 with DMEM plus 1% BSA) to triplicate 1 cm² sites of shaved,scarified skin on the back of each rabbit. Sera taken at the time ofchallenge from animals injected with L1 DNA or L1+L2 DNA containedantibody to L1 by ELISA and virus neutralizing antibody as describedabove. The animals were observed for formation of warts at 3, 6 and 10weeks following challenge. Of the rabbits that did not receive L1 DNA,51 of 54 sites challenged with CRPV developed warts, while on animalsthat received L1 DNA, 2 of 60 sites developed warts. One of the twowarts that was observed on a rabbit immunized with L1 DNA regressedwithin 3 weeks after its appearance. Table 1 shows the distribution ofwarts on rabbits after CRPV challenge. Prophylactic immunization with L1DNA protected rabbits from the development of warts upon infection withvirulent CRPV.

EXAMPLE 6

Conformational specificify of antibodies induced with L1 DNA

To demonstrate that protective neutralizing antibodies recognizeconformational epitopes on VLPs, absorption experiments were performed.Absorption of immune serum with L1 VLP (15) removed all of theneutralizing antibody and ELISA activity (FIG. 3A, B). The applicationto scarified skin of CRPV mixed with preimmune rabbit serum resulted incondylomas on all sites challenged, while when CRPV was mixed withimmune serum and similarly applied, no condylomas were seen due to theneutralizing antibody activity. When CRPV was mixed with immune serumthat had been absorbed with L1 VLPs, from which the neutralizingantibodies should have been removed, all (3/3) sites were positive forcondylomas. In contrast, when immune serum was absorbed with denaturednonparticulate L1 protein (denatured by reduction and alkylation in 8Murea), the serum was still able to neutralize CRPV (FIG. 3A), andretained its activity in the ELISA (FIG. 3B). Thus thevirus-neutralizing antibodies induced by L1 DNA immunization could beremoved only by L1 VLPs in a native conformation and not by denaturedL1. The ELISA assay appears to detect primarilyconformationally-specific antibodies reacting with intact L1 VLP, as thedepletion of ELISA activity by absorption corresponded to the removal ofneutralizing antibodies (FIG. 3B).

EXAMPLE 7

Antibody responses induced with E2 and E7 DNA

Groups of 4 NZW rabbits were injected intramuscularly with 1 mg ofV1J-E2 or V1J-E7 DNA per immunization. Four immunizations were given at0, 4, 9 and 20 weeks and were bled at 22 weeks. Antibodies were used assurrogate markers for expression of the encoded proteins. Serumantibodies were assayed using ELISA plates (NUNC Maxisorp) coated with 1μg per well of GST-E2 or GST-E7 fusion protein purified from E. colithat had been transformed with a pGEX expression vector encoding CRPV E2or E7 and induced with IPTG. The ELISA assay was performed as describedin Example 3. The net reaction rated (post dose 4 minus preimmune) inmOD/min are shown in FIG. 4. Net rates>10 mOD/min are consideredpositive; specimens with high antibody titers may have low net reactionrates at the lowest dilution due to oversaturation of the detectionsystem. Thus the encoded E2 and E7 proteins were expressed andrecognized by the recipient immune system.

    __________________________________________________________________________    SEQUENCE LISTING    (1) GENERAL INFORMATION:    (iii) NUMBER OF SEQUENCES: 14    (2) INFORMATION FOR SEQ ID NO:1:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 23 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: cDNA    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: NO    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:    CTATATAAGCAGAGCTCGTTTAG23    (2) INFORMATION FOR SEQ ID NO:2:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 30 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: cDNA    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: NO    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:    GTAGCAAAGATCTAAGGACGGTGACTGCAG30    (2) INFORMATION FOR SEQ ID NO:3:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 39 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: cDNA    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: NO    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:    GTATGTGTCTGAAAATGAGCGTGGAGATTGGGCTCGCAC39    (2) INFORMATION FOR SEQ ID NO:4:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 39 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: cDNA    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: YES    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:    GTGCGAGCCCAATCTCCACGCTCATTTTCAGACACATAC39    (2) INFORMATION FOR SEQ ID NO:5:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 39 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: cDNA    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: NO    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:    GGTACAGGATCCACCATGGCAGTGTGGCTGTCTACGCAG39    (2) INFORMATION FOR SEQ ID NO:6:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 38 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: cDNA    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: YES    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:    CCACATGGATCCTTAAGTACGTCTCTTGCGTTTAGATG38    (2) INFORMATION FOR SEQ ID NO:7:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 39 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: cDNA    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: NO    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:    GGTACAGGATCCACCATGGTTGCACGGTCACGAAAACGC39    (2) INFORMATION FOR SEQ ID NO:8:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 36 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: cDNA    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: YES    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:    CCACATGGATCCTTATTCTGCGTAGACAGCCACACT36    (2) INFORMATION FOR SEQ ID NO:9:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 39 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: cDNA    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: NO    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:    GGTACAAGATCTACCATGGAGGCTCTCAGCCAGCGCTTA39    (2) INFORMATION FOR SEQ ID NO:10:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 42 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: cDNA    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: YES    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:    CCACATAGATCTCTAAAGCCCATAAAAATTCCCTAAAAACAC42    (2) INFORMATION FOR SEQ ID NO:11:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 40 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: cDNA    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: NO    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:    GGTACAAGATCTACCATGAGCCATGGACATTGCAGGATAC40    (2) INFORMATION FOR SEQ ID NO:12:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 39 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: cDNA    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: YES    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:    CCACATAGATCTTTATAAGCTCGCGAAGCCGTCTATTCC39    (2) INFORMATION FOR SEQ ID NO:13:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 41 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: cDNA    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: NO    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:    GGTACAAGATCTACCATGATAGGCAGAACTCCTAAGCTTAG41    (2) INFORMATION FOR SEQ ID NO:14:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 36 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: cDNA    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: YES    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:    CCACATAGATCTTCAGTTACAACACTCCGGGCACAC36    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What is claimed is:
 1. A papillomavirus (PV) vaccine for use in a humancomprising a vector comprising:(a) a polynucleotide encoding at leastone human papillomavirus (HPV) gene from an HPV selected from the groupconsisting of: HPV 6a, HPV 6b, HPV 11, HPV 16 and HPV 18, and expressinga protein selected from the group consisting of: L1 and L1+L2, (b) a CMVpromoter for RNA polymerase transcription; (c) a transcriptionalterminator from a bovine growth hormone gene; and (d) a neomycinresistance marker gene;wherein the vector is present in aphysiologically acceptable solution.
 2. A vaccine according to claim 1wherein the vector is V1Jneo or V1Jns.
 3. A method for inducing animmune response to HPV in vivo comprising introducing into a livingtissue of a human an HPV vaccine wherein said vaccine comprises a vectorcomprising:(a) a polynucleotide encoding at least one HPV L1 gene froman HPV virus selected from the group consisting of HPV 6a, HPV 6b, HPV11, HPV 16, and HPV 18; (b) a CMV promoter for RNA polymerasetranscription; (c) a bovine growth hormone transcriptional terminator;and (d) a neomycin resistance marker gene;wherein the vector is presentin a physiologically acceptable solution and wherein said L1 gene isexpressed and an immune response is induced.
 4. A method according toclaim 3 wherein the vector is V1Jneo or V1Jns.